| The complex in vivo barriers severely affect the targeted antitumor chemo-therapeutics delivery efficiency of nano-drug delivery systems(NDDSs).Ideal NDDSs show strong antitumor efficiency only after the long blood circulation,the accumulation and deep penetration in tumor tissues,the efficient cellular internalization and the responsive intracellular drug release,while the physiochemical properties of NDDSs,like size,surface charge,PEG density and the surface functionalities,affect the in vivo fate and final biological effects of nanomedicine.The different in vivo barriers require for different features of NDDSs,thus paradox existed.Surface charge-reversible and size-reversible NDDSs were developed based on the rationale of size and surface charge effects on the biological processes.However,little is known about the effects of hydrophobicity/hydrophilicity of NDDSs on their in vivo processes and biological effects.It was reported that hydrophilic nanomaterials favored long blood circulation by avoiding the protein adsorption in blood and phagocytosis by RES(Reticuloendothelial System),while hydrophobic ones facilitated the cellular internalization by the hydrophobic interaction with the cellular membrane.Hence hydrophilicity/hydrophobicity reversible NDDSs could solve the paradox existed in the blood circulation and the cellular internalization.PEGylation increases the hydrophilicity of nanomaterials,while responsive PEG cleavage in the tumor acidic microenvironment increases the hydrophobicity,thus PEG dilemma is settled down.Nevertheless this kind of hydrophilicity/hydrophobicity reversal is based on the pH induced functional chemical bond cleavage,the time consuming process affects the final antitumor efficiency.Meanwhile the effects of the hydrophilicity/hydrophobicity of nanomaterials on the tumor deep penetration are unknown.The previous work in our group proposed that the hydrophilicity/hydrophobicity reversal of nanogels in tumor acidic microenvironment promoted the cellular uptake by tumor cells.Nanogels are nano-sized hydrophilic network containing large amount of water molecules,while the hydrophobic transition from hydrophilic state based on the protonation of the backbone structure groups is expeditious.The hydrophilicity/hydrophobicity reversible nanogels provide new horizons for the development of tumor microenvironment responsive NDDSs.This thesis is based on pH-regulated hydrophilicity/hydrophobicity reversal nanogels,the effects on blood circulation,accumulation in tumor sites and the deep penetration in tumor tissues have been evaluated.The nanogels were also endued with pH regulated surface charge reversal and redox responsiveness,in order to enhance the antitumor efficiency of the DOX loaded nanogels by promoting the lysosome escape and intracellular GSH responsive drug release.The main contents and results are as following,1)pH regulated hydrophilicity/hydrophobicity reversible-and charge reversible-PNSM nanogels were synthesized by modulating the ratios among temperature sensitive monomer N-isopropyl acrylamide(NIPAM),pH sensitive monomer N-Methyallyl amine(MAA)and zwitterionic monomer sulfobetaine methacrylate(SBMA)(denoted as R-nanogels).PNSM nanogels were crosslinked by N,N'-bis(acryloyl)cystamine(BAC),the disulfide bond existed in the crosslinker endowed the redox responsiveness for the nanogels.By modulating the ratios among NIPAM,MAA and SBMA,PNSM nanogels without pH regulated hydrophilicity/hydrophobicity reversibility were also synthesized(denoted as C-nanogels).The transmittance measurements and size analysis methods confirmed that R-nanogels were hydrophilic in physiological blood and normal tissues(pH 7.4,37 ?),while they spontaneously turned to hydrophobic in the acidic tumor microenvironment(pH 6.5,37 ?).C-nanogels kept hydrophilic at pH 7.4 and 6.5.R-nanogels showed charge reversal at pH 4.5,suggesting the proton sponge effect in lysosome.DOX was loaded into the PNSM nanogels according to the solvent evaporation method,and the release profile showed pH and GSH dual responsiveness of R-nanogels.2)The adsorption of fetal bovine serum(FBS)and phagocytosis by macrophage Raw264.7 at pH 7.4 and 6.5 were compared,the results showed that pH regulated hydrophilicity/hydrophobicity reversibility of R-nanogels avoided the protein adsorption and phagocytosis by macrophage Raw264.7 at pH 7.4.The pharmacokinetics study suggested long blood circulation for both DOX-loaded C-nanogels and R-nanogels.pH regulated hydrophilicity/hydrophobicity reversibility also benefited the internalization by hepatocarcinoma cells HepG2 and H22 cancer stem cells at pH 6.5.Which would resolve the paradox existed in the blood circulation and cellular internalization.To confirm the intracellular GSH responsive drug release for DOX-loaded R-nanogels,glutathione monoethylester(GSH-Oet)was applied,the results suggested that GSH-Oet pre-treatment increased the intracellular DOX fluorescence and the cytotoxicity against HepG2 cells of DOX-loaded R-nanogels.The colocalization of DOX-loaded R-nanogels with intracellular organisms was also analyzed,and the results showed that pH regulated charge reversibility favored the lysosome escape,while the intracellular GSH responsiveness helped the DOX release into nucleus.3)The distribution of DOX-loaded PNSM nanogels in xenograft mice was studied,the results showed that the tumor accumulation content of DOX-loaded R-nanogels was significantly higher than that of DOX-loaded C-nanogels and free DOX,meanwhile DOX-loaded R-nanogels showed enhanced retention in tumor tissue.The penetration in tumor tissues is the main hindrance for the antitumor efficiency of NDDSs,thus the tumor tissue penetration of DOX-loaded PNSM nanogels was also explored.The hydrophilicity/hydrophobicity reversibility of DOX-loaded R-nanogels in the acidic microenvironment promoted the penetration in H22 tumor spheroid,ex vivo tumor and in vivo tumor models.4)The antitumor study of DOX-loaded PNSM nanogels suggested that DOX-loaded R-nanogels showed significantly higher antitumor efficiency in H22 exnograft mice compared with DOX-loaded C-nanogels and free DOX.DOX-loaded R-nanogels could increase the tumor cells apoptosis and inhibit the tumor cell proliferation.Tumor stem cells in the deep hypoxic area are related with the tumor recurrence and metastasis,so the effects of DOX-loaded PNSM nanogels on cancer stem cell were also studied,the results showed stronger cytotoxicity of DOX-loaded R-nanogels on the cancer stem cells from the exnograft mice.PNSM nanogels could decrease the cardiotoxicity of the payload with good safety.5)To further confirm the efficiency and university of pH regulated hydrophilicity/hydrophobicity reversal strategy,the temperature sensitive monomer oligoethyleneglycol with good biocompatibility,and pH sensitive acrylic acid were crosslinked with BAC to achieve the pH regulated hydrophilicity/hydrophobicity reversal and redox responsive P(OEGMAs-ss-AA)nanogels.The effects of pH regulated hydrophilicity/hydrophobicity reversibility on protein adsorption and cellular internalization by cancer cells were studied,the results showed that the hydrophilicity/hydrophobicity reversibility of P(OEGMAs-ss-AA)nanogels in the tumor acidic microenvironment could solve the paradox existed in the blood circulation and cellular internalization.We also analyzed the intracellular fate of DOX-loaded P(OEGMAs-ss-AA)nanogels,the results showed that the DOX-loaded nanogels entered into Bel7402 cells by caveolin-and clathrin-dependent endocytosis together,and release the DOX into nucleus from lysosome after the trafficking in early endosomes and late endosomes.In summary,pH induced hydrophilicity/hydrophobicity reversible-nanogels were hydrophilic and swollen in blood and normal tissues,which avoided the protein adsorption in blood and opsonization by RES,thus favored the blood long circulation.They reversed to hydrophobic and shrunken in the tumor acidic microenvironment,which benefitted the internalization into tumor cells and cancer stem cells.Such nanogels resolved the paradox existed in blood circulation and cellular internalization.The results also suggested that pH induced hydrophilic/hydrophobic reversibility of the nanogels contributed to the accumulation and deep penetration in tumor tissues.On the other hand,pH-responsive surface charge reversal of the nanogels makes the surface charge shifted from negative to positive in the acidic environment of lysosome after cellular internali-zation,which was propitious to the lysosome escape to the cytoplasm.The disulfide bond in nanogels endowed the redox-responsive intracellular drug release property.The pH-responsive hydrophilicity/hydrophobicity and surface charge-reversible and redox responsive nanogels showed excellent antitumor efficiency and the pH-responsive hydrophilicity/hydrophobicity reversible nanogels provide new direction for exploring microenvironment induced reversal nano-drug delivery systems. |
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